AAT Bioquest

What is the difference between immunofluorescence, immunohistochemistry and immunocytochemistry?

Posted February 25, 2020


All three techniques are similar in that they use antibodies to selectively identify targets of interest in cells or biological tissue sections, either directly or indirectly. In direct detection methods, a single primary antibody is conjugated to a detectable tag, such as an enzyme or fluorophore, and is used in a single-step procedure to directly detect the target of interest. With indirect detection, two antibodies are used in sequence for the detection of a target antigen. First, the sample is incubated with an unlabeled primary antibody directed against the target antigen. Then, a labeled secondary antibody specific for the primary antibody is used to detect its presence, and thus the target of interest.

Generally speaking, for antibodies conjugated to an enzyme (e.g. HRP) visualizing the antibody-antigen interaction requires a substrate specific to the enzyme tag being used. The enzyme catalyzes a color-producing reaction with its respective substrate, and the resulting chromogenic signal can then be detected using either a spectrophotometer or an absorbance microplate reader. For antibodies conjugated to a fluorophore, detect using a fluorescence instrument (e.g. fluorescence microscope, fluorescence microplate reader or flow cytometer).

The three staining techniques differ in the sample/tissue type:

  • immunofluorescence is commonly used to stain microbiological cells
  • immunohistochemistry is commonly used to stain sections of biological tissue
  • immunocytochemistry is commonly used to stain intact cells removed from extracellular matrix
Additional resources

Secondary Antibodies

Horseradish Peroxidase (HRP) and Poly-HRP

Immunohistochemistry (IHC)